7. Project Summary: Melanin-concentrating hormone is an neuropeptide produced primarily in the lateral hypothalamic area of the brain that potently increases appetite, food intake, and body weight 1,2. Importantly, chronic central pharmacological blockade of MCH receptors (MCH1Rs) reverses diet-induced obesity in mice 3,4 and therefore there is recent interest in developing obesity pharmacotherapies targeting the MCH system 5-8. While MCH-producing neurons extensively project throughout the brain 9 and central MCH1Rs are widely distributed 10, very little is presently known about the neuronal pathways and behavioral mechanisms mediating the potent orexigenic effects of MCH. Our preliminary data reveal that central MCH signaling in rats increases both normal chow intake as well as conditioned reward-based feeding behaviors, including impulsive responding and conditioned place preference for palatable food. We further identify two novel signaling targets through which MCH neurons promote orexigenic effects: [1] ?bulk flow? signaling through the cerebral ventricles following MCH release into the cerebral spinal fluid (CSF), and [2] synaptic signaling to the nucleus accumbens shell (ACBsh), a brain substrate critically associated with learned aspects of food reward 11,12. We hypothesize that MCH CSF- and ACBsh-signaling pathways differentially regulate normal vs. reward-based feeding, respectively. This hypothesis is supported by our preliminary data showing that chemogenetic activation of ACBsh-projecting MCH neurons increases palatable food (sucrose, high fat diet) intake without affecting intake of bland chow, whereas CSF MCH injections equally increase intake of a less- and more- preferred food (chow vs. sucrose), and endogenous MCH CSF levels are elevated prior to regular nocturnal chow intake. Our hypothesis will be examined in Aim 1, where we investigate the effects of chemogenetic activation of specific MCH neuronal populations that project to either the CSF or the ACBsh on various feeding behaviors (e.g., habitual, circadian, conditioned reward-based). Conditional virus-based neural pathway tracing strategies are used in Aim 2 in order to identify the collateral projections of CSF- and MCH-projecting MCH neurons, as well as the 2nd-order targets of MCH neurons that receive input from the medial prefrontal cortex and the basolateral amygdala, two brain regions that are critically involved in reward-based feeding 13. In conjunction with retrograde neural pathway tracing, neurochemical phenotyping of these populations of MCH neurons will be done using fluorescence in situ hybridization and immunohistochemistry techniques. Finally, Aim 3 utilizes a [14C]-iodoantipyrine-based autoradiographic brain mapping method to identify functional downstream neural systems through which MCH neurons elevate feeding. Results will reveal brain networks engaged by chemogenetic activation of ACBsh-projecting MCH neurons at resting state, during intake of palatable sucrose, and during presentation of a sucrose-conditioned cue. Neural networks engaged by activating CSF-projecting MCH neurons will also be identified, at resting state and during chow consumption. Overall, results from this proposal will identify the neurobiological pathways and behavioral mechanisms whereby MCH engages normal and reward-based feeding, thus contributing critical insight into feeding behavior, and advancing toward strategies to reverse excessive feeding.